1. Field of the Invention
This invention relates to a pullulanase-like enzyme, a method for the preparation thereof, and a method for the saccharification of starch with the enzyme.
2. Description of the Prior Art
Pullulanase is an enzyme which cleaves the .alpha.-1,6-glucosidic linkages of pullulan and eventually produces maltotriose. It is known to be produced by various bacteria and streptomyces. It is known that the pullulanase, when left acting upon amylopectin or starch containing amylopectin, cleaves the branched linkages (.alpha.-1,6-glucosidic linkages) thereof and gives rise to an amylose-like polysaccharide and, therefore, causes increase of the iodine reaction. It is, however, incapable of cleaving the .alpha.-1,4-glucosidic linkages. In contrast, amylases such as .alpha.-amylase and .beta.-amylase which cleave the .alpha.-1,4-glucosidic linkages are incapable of cleaving the .alpha.-1,6-glucosidic linkages. As a very rare exception, glucomaylase is capable of cleaving not only the .alpha.-1,4-glucosidic linkages but also the .alpha.-1,6-glucosidic linkages.
In the enzymes which cleave the .alpha.-1,6-glucosidic linkages, there are included, besides pullulanase, various enzymes called isoamylase, R-enzyme, and amylo-1,6-glucosidase. These enzymes are collectively referred to as .alpha.-1,6-glucosidases or, more commonly, as debranching enzymes.
Recently, these debranching enzymes including pullulanase are used in conjunction with .beta.-amylases to product maltose from starch in high yields through their cooperation on the starch. With a view to making upon for glucoamylases' insufficient ability to cleave branches (.alpha.-1,6-glucosidic linkages), they are also used in conjunction with such glucoamylases so as to permit production of glucose from starch in high yields. Thus, the debranching enzymes are useful even for the production of glucose.
For pullulanase to be effectively used jointly with glucoamylase, for instance, since glucoamylase optimally works in a pH range of 4 to 5 and a temperature range of 55.degree. to 60.degree. C., the pullulanase has to possess normal thermal stability to withstand the temperatures of 55.degree. to 60.degree. C. for a long time and work effectively at pH 4 to 5.
Unfortunately, numerous debranching enzymes heretofore known to the art, with the exception of a few of microorganic origins (Bacillus stearothermophilus (Collection of Summaries of Lectures for the 1972 Annual Meeting of Japan Agriculture Chemical Society, page 88, optimum temperature 65.degree. to 67.5.degree. C.) and Bacillus acidopulluliticus (Japanese Patent Public Disclosure SHO 57(1982)-174089; Starch 34, 340 (1982), optimum temperature 60.degree. C.)), have their optimum temperatures in the neighborhood of 40.degree. to 50.degree. C., thus suffering from inferior thermal stability.
An object of this invention is to provide a thermostable pullulanase-like enzyme possessing the optimum pH in the range of about 5 to about 7.5.
Another object of this invention is to provide the enzyme capable of producing maltose and maltotriose in high yields.
A further object of this invention is to provide a method for promoting the saccharification of starch by glucoamylase and therefore enhancing the yield of glucose.